Effect of Nitrogen, Air, and Oxygen on the Kinetic Stability of NAD(P)H Oxidase Exposed to a Gas–Liquid Interface

Biocatalytic oxidation is an interesting prospect for the selective synthesis of active pharmaceutical intermediates. Bubbling air or oxygen is considered as an efficient method to increase the gas–liquid interface and thereby enhance oxygen transfer. However, the enzyme is deactivated in this process and needs to be further studied and understood to accelerate the implementation of oxidative biocatalysis in larger production processes. This paper reports data on the stability of NAD(P)H oxidase (NOX) when exposed to different gas–liquid interfaces introduced by N2 (0% oxygen), air (21% oxygen), and O2 (100% oxygen) in a bubble column. A pH increase was observed during gas bubbling, with the highest increase occurring under air bubbling from 6.28 to 7.40 after 60 h at a gas flow rate of 0.15 L min–1. The kinetic stability of NOX was studied under N2, air, and O2 bubbling by measuring the residual activity, the deactivation constants (kd1) were 0.2972, 0.0244, and 0.0346 with the corresponding half-lives of 2.2, 28.6, and 20.2 h, respectively. A decrease in protein concentration of the NOX solution was also observed and was attributed to likely enzyme aggregation at the gas–liquid interface. Most aggregation occurred at the air–water interface and decreased greatly from 100 to 14.16% after 60 h of bubbling air. Furthermore, the effect of the gas–liquid interface and the dissolved gas on the NOX deactivation process was also studied by bubbling N2 and O2 alternately. It was found that the N2–water interface and O2–water interface both had minor effects on the protein concentration decrease compared with the air–water interface, whilst the dissolved N2 in water caused serious deactivation of NOX. This was attributed not only to the NOX unfolding and aggregation at the interface but also to the N2 occupying the oxygen channel of the enzyme and the resultant inaccessibility of dissolved O2 to the active site of NOX. These results shed light on the enzyme deactivation process and might further inspire bioreactor operation and enzyme engineering to improve biocatalyst performance.


■ INTRODUCTION
−3 For example, alcohol dehydrogenase (ADH) has been used to oxidize lactols to chiral intermediate lactones together with NAD(P)H oxidase (NOX) for cofactor regeneration on a 100 L scale. 4 Likewise, a renewable and sustainable route for polymer production based on 2,5-furandicarboxylic acid (FDCA) can be realized via the oxidation of biobased 5-hydroxymethylfurfural (HMF) using galactose oxidase (GOase), 5,6 which would be an ideal way to utilize renewable biomass for sustainability. 7Generally, monooxygenases and oxidases have been developed for biocatalytic oxidation processes, and oxygen has been supplied as an electron acceptor (oxidant) with the ensuing H 2 O or H 2 O 2 -associated byproducts. 8Additionally, peroxygenases and dioxygenases have also been developed (although to a lesser extent), which makes the toolbox of the biocatalytic oxidation process even more diverse, but enzyme engineering is still required for enzyme activity and stability improvements and developing a broad substrate scope. 9,10However, dehydrogenases are still attractive and relatively well developed with enhanced activity and thermostability. 11They are routinely used for reduction but can also be applied in biocatalytic oxidation processes together with the nicotinamide cofactor NAD(P) + as an electron acceptor.Likewise, (R)-undecavertol (>98%, ee) was produced by (S)-selective ADH alcohol dehydrogenase with a 400 g L −1 substrate concentration on a pilot scale. 12Meanwhile, the cofactor NAD(P) + can be regenerated in situ by NOX for economic sustainability. 13uring this reaction, oxygen is generally considered as the most suitable oxidant since it is economical and readily available in air, with water as the only byproduct (Scheme 1).
Such a biocatalytic oxidation route would seem to be an ideal process for industry, the oxygen from air is the ideal oxidant for the biocatalytic oxidation process, while pure oxygen can be supplied to promote the oxygen mass transfer in industries where this is allowed. 4,13However, the effect of pure oxygen in the gas phase on enzyme stability is not known and requires further study.In previous work, it was found that the ADH/NOX was more stable with a low oxygen gas flow rate and resulted in a high product conversion to produce (R)undecavertol with a pure oxygen supply, achieving a space− time yield of 14 g L −1 h −1 . 12In another report, it was found that the stability of L-amino acid oxidase was affected by the dissolved oxygen concentration and that the reaction rate and productivity were increased by fast and continuous bubbling with air. 14In addition, it turns out that the affinity constant of an oxidase toward oxygen (K MO ) is also an important factor that determines the way the oxygen should be supplied. 5,15,16If the K MO of an enzyme is low, air supply might be adequate to reach the maximum activity, and the dissolved oxygen is not limiting. 17For example, the initial reaction rate of a lipoxygenase to convert linoleic acid into linoleic acid hydroperoxide has no significant decrease when the initial dissolved oxygen concentration was adjusted from 10 to 80% of saturation, which indicates a low K MO for lipoxygenase. 18herefore, the increasing oxygen concentration may not result in high enzyme activity if the K MO of an oxidase is low, and the high activity and stability can be achieved through protein engineering alone. 19,20However, a gas phase with high oxygen concentration may decrease the reaction rate because of fast enzyme deactivation. 21It was also reported that hydrogenases are sensitive to oxygen concentration since their activity decreases as the oxygen partial pressure increases. 22,23herefore, an oxidant with air or pure oxygen is a critical issue that may have different effects on enzyme activity and stability.Further study is required to understand and guide the application of biocatalytic oxidation in industry.
In one study, in a controlled bubble column, we previously reported that NOX deactivation was related to the residence time of the gas−liquid interface, the long half-life of NOX under fast gas bubbling was attributed to the low residence time of bubbles in the bubble column. 13On this basis, we herein further investigate the effect of gas on the kinetic stability of NOX when bubbling different gases.The effect of N 2 , air, and O 2 on NOX stability was studied by measuring the residual activity of NOX after different gas bubbling times.The operation conditions of a running bubble column (pH, temperature, and dissolved O 2 concentration) were also measured online by relevant sensors.The protein concentration of NOX solution was also evaluated during the experiments, and the corresponding specific activity of NOX was calculated to further understand and distinguish the effect of the dissolved gas and gas−liquid interface on the kinetic deactivation.Structural analysis of NOX was also carried out to help explain the NOX deactivation process at gas−liquid interface.This study was intended to help understand the enzyme deactivation process at a gas−liquid interface and provide inspiration for protein engineering to further improve the NOX performance under process conditions.

Organic Process Research & Development
Sigma-Aldrich (St. Louis, MO).The Coomassie Plus protein assay reagent was purchased from Thermo Fisher Scientific (Waltham, MA).All chemical reagents used in experiments were of analytical grade and used directly without any further purification.
Experimental Procedure.Enzyme Activity Assay.The activity assay and kinetic stability experiments for NOX have been described in our previous study. 13In detail, a NOX solution (500 μL, 0.05 g L −1 ) was mixed together with NADPH (1 g L −1 , 100 μL) and phosphate buffer (50 mM, pH 7.0, 400 μL) in a semi-micro cuvette, which was shaken using a mixer (ZX3 Advanced Vortex Mixer, Usmate Velate, Italy) at 1600 rpm at 25 °C.Then, the sample was measured by a UV− vis spectrophotometer (Shimadzu UV-1800, Kyoto, Japan) for 2 min, and the decreasing rate of NADPH adsorption at 340 nm was correlated with the activity of NOX.The corresponding extinction coefficient was ε 340 = 6.22 mM −1 cm −1 .One unit (U) of NOX activity was defined as 1 mM NADP + produced per minute at 25 °C, pH 7.0.The liquid level of NOX solution in bubble column was marked and replenished with deionized water in time due to the water evaporation caused by the continuous gas bubbling.
Kinetic Stability of NOX.A NOX solution (200 mL, 0.05 g L −1 ) was incubated in a bubble column to study the kinetic stability under various gases and rates of bubbling (Figure 1).The gas flow rate was controlled by an oxygen flow meter and a nitrogen flow meter (Sierra SmartTrak 50, Monterey, CA) together with SmartTrak 50 product software (S50 firmware, version 1.05).Samples were taken during gas bubbling to measure the residual activity of NOX (Figure S1).The protein concentration was measured by the Coomassie Plus protein assay reagent at a ratio of 1:1 with NOX solution and measured at 595 nm using a UV−vis spectrophotometer (Figure S2).
Bubble Column Data Collection.In our previous work, the gas bubble shape and the area of the gas−liquid interface have been described. 13Here, the pH, temperature, and dissolved O 2 concentration in the enzyme solution of the bubble column were monitored online by the Pyro Workbench connected to sensors FireSting-PRO (4 Channels, PyroScience GmbH, Aachen, Germany).The pH sensor was calibrated with the standard solution at pH 2 and 11 before use.The pH values of deionized water and the NOX solution before and after gas bubbling were measured by a PHM220 MeterLab pH meter (Radiometer Analytical, Lyon, France).The pH meter was calibrated with a standard solution at pH 7 and 10 before use.
Calculation Methods.The residual activity of NOX was fitted with a first-order deactivation kinetic experiment to calculate the deactivation kinetics where a and a 0 are the enzyme activity at t = 0 h and t h, and k d is the enzyme deactivation kinetic constant.k d1 and k d2 are also introduced to represent the non-first-order degradation over the entire degradation.The two constants represent the relevant deactivation constants for the first and second stages.
The dissolved gas concentration of N 2 , air, and O 2 are calculated by Henry's law.

Organic Process Research & Development
where C is the solubility of a gas in a solvent, H is Henry's law constant, and P gas is the partial pressure of the gas.

■ RESULTS AND DISCUSSION
Online Monitoring of NOX Solution during Gas Bubbling.The concentration of dissolved O 2 is critical for most biocatalytic oxidation processes, as also the pH and temperature of incubation.Therefore, the online conditions such as temperature, pH, and dissolved O 2 sensors of NOX solution incubated in the bubble column were monitored during gas bubbling, and the results are shown in Figure 2. The temperature of the bubble column was controlled by a circular water bath for 25 °C during all experiments.The equilibrium between dissolved O 2 and the O 2 in the gas phase increased quickly according to the results in Figure 2, and likewise, the dissolved O 2 could be removed very quickly and finally reached zero with N 2 bubbling (Figure 2a).With gas bubbling, the dissolved O 2 concentration was maintained constant at 0, 21, and 100% with N 2 , air, and O 2 bubbling, respectively (Figure 2a−c).The air-saturated water was a 21% O 2 concentration in the gas phase that coincided with the dissolved O 2 concentration of 0.26 mM.The pH increase of NOX solution was also found during gas bubbling.In Figure 2a, the pH increased from 6.04 to 6.38 after 6 h of N 2 bubbling.With O 2 bubbling, it increased from 6.12 to 7.02 after 60 h of O 2 bubbling.The dissolved O 2 concentration was 1.24 mM, where the dissolved O 2 concentration was 5-fold higher than that in air-saturated water (Figure 2c).It is worth mentioning that the highest pH increase was observed with air bubbling, and it increased from 6.28 to 7.40 after 60 h of bubbling with 0.15 L min −1 gas flow rate (Figure 2b).As a control experiment (Figure 2d), the pH of deionized water was little changed after 60 h of gas bubbling with the same gas flow rate of 0.15 L min −1 .Therefore, the pH increase of NOX solution during gas bubbling might only be attributed to the deactivation of NOX.It has been reported that the bubbles in deionized water were negatively charged, and the gas−liquid interface charge was independent of the bubbling gas. 24Therefore, the pH change was only caused by the NOX deactivation process, and the difference of pH change in Figure 2a−c was attributed to the effect of different gas−liquid interfaces on the enzyme deactivation process.Meanwhile, the enzyme might be regarded as a surfactant and contained both hydrophilic and hydrophobic residues, and therefore was adsorbed at the gas− liquid interface.The increasing pH was also related to a decreasing ζ potential of the gas−liquid interface, which might also relate to enzyme unfolding and then its adsorbing at the interface.
NOX Kinetic Stability under N 2 , Air, and O 2 Bubbling.Effect of Different Gases.In another experiment, the NOX kinetic stability was investigated in a bubble column under 0.05 L min −1 N 2 , air, or O 2 gas bubbling with 200 mL of 0.05 g L −1 NOX solution at 25 °C (Figure 3).A schematic diagram of the gas−liquid interface (Figure 3a) illustrates the continuously and constantly generated gas−liquid interface by gas bubbling, which promotes the mass transfer of oxygen from the gas to the liquid phase effectively.With a controlled gas bubbling rate, the gas−liquid interfacial area was easy to calculate   The ratio between the NOX concentration and the gas−liquid interface area (E ratio ) was used to describe the operating conditions in a bubble column reactor. 13The kinetic stability of NOX was studied under N 2 , air, and O 2 bubbling by measuring the residual activity; the deactivation constants were 0.2972, 0.0244, and 0.0346 (k d1 ) with the corresponding halflives of 2.2, 28.6, and 20.2 h (Table 1).Two-stage deactivation kinetics was found when NOX was exposed to the air−water interface with 0.05 L min −1 gas flow rate, and the k d1 and k d2 were 0.0244 and 0.1150, respectively.It was obvious that the deactivation constant in the second stage (k d2 ) was much higher than in the first stage (k d1 ).However, single-stage deactivation kinetics was both found when NOX was exposed to N 2 and O 2 bubbling with the same gas flow rate.The k d of N 2 bubbling was 0.2972, which was 12-fold and 9-fold higher than those with air and O 2 bubbling, respectively.It has been reported that the deactivation of NOX at the air−water interface is a time-dependent process, and a long residence time of the interface results in a more serious deactivation of NOX. 13 This implies that N 2 and O 2 themselves (dissolved N 2 and O 2 ) have effects on NOX stability other than the interfaces.The residual activity of NOX under N 2 bubbling dropped sharply from 100 to 23.6% in the first 6 h with a deactivation constant of 0.2972, which was 12-fold higher than under air bubbling (Figure 3b,c), which indicated that NOX was less stable with N 2 bubbling and N 2 was harmful to NOX stability.The NOX has the smallest deactivation constant of 0.0244 under air bubbling compared with N 2 and O 2 bubbling, and the residual activity decreased at an almost nearly constant rate during the experiment (Table 1 and Figure 3c).The NOX deactivation constant under O 2 bubbling was 0.0346, which was bigger than under air bubbling (Figure 3d).This result might be caused by the high O 2 concentration of dissolved O 2 and also the O 2 −water interface where the amino acid residues of NOX were oxidized, resulting in faster deactivation than under air bubbling. 25However, the deactivation of NOX under N 2 bubbling needed further study because N 2 is generally considered a nonreactive gas.Two possible factors (the N 2 − water interface and the dissolved N 2 in water) might be responsible for this result.In the following experiments, the different effects of dissolved gas and the gas−liquid interface on NOX stability were further verified by bubbling N 2 and O 2 alternately.
Additionally, the effect of argon on NOX stability was investigated and compared with N 2 bubbling.The results are shown in Figure 4.The deactivation of NOX under argon bubbling was also fast and had a similar decreasing trend to N 2 bubbling, and the half-life under argon and N 2 bubbling were 1.14 and 2.2 h, respectively (Figure 4a,b).The deactivation constant k d of argon bubbling was 2-fold higher than that for N 2 bubbling (0.5715 vs 0.2817), suggesting that NOX deactivation occurred more rapidly under argon bubbling (Figure 4c,d).Notably, NOX was much more stable under air bubbling (half-life of 28.6 h) compared with argon and N 2 bubbling (Figure 3c).

Organic Process Research & Development
Effect of Dissolved Gas and the Gas−Liquid Interface.When the enzyme solution was bubbled with gas, the enzyme might be affected by two factors; first, the gas−liquid interface introduced by gas bubbling, and second, the dissolved gas in the aqueous solution (as a result of the mass transfer and phase equilibrium between gas and liquid phases) (Figure 5a).The change of gas phase composition affected the enzyme activity and stability through these two factors.These two effects were further studied in these experiments by first N 2 bubbling for 6.5 h and then O 2 bubbling (Figure 5b), and also the inverse way by first O 2 bubbling for 6.5 h and then N 2 bubbling (Figure 5c).As shown in Figure 5b, the residual activity of NOX decreased sharply from 100 to 23.3%, with N 2 bubbling for the first 6.5 h.Subsequently, the NOX activity was not recovered with the following O 2 bubbling, which indicated that this deactivation of NOX caused by N 2 bubbling was irreversible (probably because the gas channel of NOX was fully occupied by the dissolved N 2 first and the following dissolved O 2 could not enter the active site of NOX anymore).Furthermore, this phenomenon might also be observed in other enzymes containing a gas channel and needing gas as a substrate (e.g., glucose oxidase, galactose oxidase, lipoxygenase).Potentially, if the gas channel was occupied by another nonsubstrate gas, the enzyme activity would lose rapidly and irreversibly.It has been reported that the oxidation performance of oxygenase could be improved by oxygen channel engineering. 20In Figure 5c, NOX was first bubbled with O 2 for 6.5 h, and the NOX activity decreased from 100 to 85%.Following N 2 bubbling, the NOX activity decreased slowly from 85 to 14% after 28 h, and the activity loss was notably slower compared with Figure 5b, which might indicate that the oxygen channel of NOX was first occupied and saturated by O 2 ; therefore, the dissolved N 2 would not take over the active site of NOX, and only the N 2 −water interface affected the deactivation of NOX.
In Figure 5b,c, it can be concluded that the dissolved N 2 had a significant effect on NOX activity compared with the N 2 − water interface.The deactivation rate of NOX residual activity in Figure 5c when N 2 was bubbled (after 6.5 h) was lower than that of the first 6.5 h of N 2 bubbling in Figure 5b.In addition, both dissolved O 2 and the O 2 −water interface had a small effect on NOX activity from Figures 3b and 5c.Therefore, the gas channel should be carefully considered since it may be very sensitive and might lose activity when nonsubstrate gases are present.
Furthermore, the dissolved N 2 and O 2 concentrations were calculated by Henry's law, and the results are listed in Table 1.With N 2 bubbling, NOX was affected by the N 2 −water interface and the dissolved N 2 (0.65 mM, 25 °C, 1 atm) at the same time.In contrast, NOX was affected by the O 2 −water interface and dissolved O 2 (1.24 mM, 25 °C, 1 atm).With air or N 2 bubbling, the dissolved N 2 concentration was always higher than the dissolved O 2 concentration.Oxygen might have the advantage of preferentially diffusing into the oxygen channels. 26,27According to these results, Figure 5b,c, the dissolved N 2 was more harmful to NOX activity compared with dissolved O 2 .
Protein Concentration Change during Bubbling.The protein concentration of the NOX solution was measured under quiescent conditions and also gas bubbling, resulting in a decrease during gas bubbling (Tables S1−S4).The result is shown in Figure 6a.Under quiescent conditions, there was no decrease of protein concentration, but the specific activity of NOX decreased as expected (Figure 6a,b).The fitting equation of the NOX specific activity decrease was y = −0.079x+ 14.361 (R 2 = 0.95), and it was related to the natural deactivation of NOX in solution.In a bubble column, it was clear that the protein concentration decreased gradually under exposure to the gas−liquid interface.The fastest decrease of protein concentration occurred with air bubbling from 100 to 14% after 60 h with 0.15 L min −1 gas flow rate at 25 °C (Figure 6a).With N 2 and O 2 bubbling, the protein concentration decreased from 100 to 68% and 65%, respectively.
However, the specific activity of NOX decreased the fastest over a short N 2 bubbling time compared with air bubbling and O 2 bubbling (Figure 6c−e).The corresponding fitting equation was y = −1.644x+ 13.111 (R 2 = 0.94), where the decreasing slope was 21-fold, 16-fold, and 10-fold faster than quiescent conditions, air bubbling, and O 2 bubbling.This result indicated that the dissolved N 2 caused NOX deactivation rapidly and further confirmed that N 2 might occupy the oxygen channel of NOX.This NOX with less activity (or total loss of activity) was still dissolved in solution and resulted in a similar protein concentration decrease compared with O 2 bubbling (Figure 6a).However, decreasing NOX specific activity with O 2 bubbling might be mainly caused by the over-oxidation of the NOX amino acid residue because the dissolved O 2 concentration was much higher compared with air-saturated solution. 28The fitting equation of the specific activity decrease with O 2 bubbling was y = −0.171x+ 17.493 (R 2 = 0.83) (Figure 6e).It should also be mentioned that the impurity protein in NOX solution might also be responsible for the protein concentration decreasing, which could also affect the specific activity.
In addition, the decreasing slopes of the NOX specific activity under quiescent conditions and air bubbling had a similar trend and were 0.079 and 0.106, respectively (Figure 6b,d), but the protein concentration under air bubbling decreased fast.It might indicate that the air−water interface only caused protein aggregation, and then the protein aggregation formed an insoluble precipitate and left the aqueous solution.The dissolved gases (N 2 and O 2 ) at this concentration had a limited effect on enzyme activity.It might also be the interface that caused a large decrease in NOX activity and meanwhile led to a decrease in the protein concentration, which also showed a similar decrease of the NOX specific activity under quiescent conditions and air bubbling (Table S5).Nevertheless, it was still suggested that O 2 was more suitable for gas bubbling to the bubble column for NOX-catalyzed cofactor regeneration because of the high total activity of NOX under O 2 bubbling during 60 h incubation.However, NOX was always used in cascade reactions together with dehydrogenase, and the stability of these dehydrogenases and cofactors has rarely been studied.Though the oxygen mass transfer coefficient k L a would be increased with pure O 2 bubbling, most of these dehydrogenases dissolved in solution might be sensitive to high concentrations of dissolved O 2 , which might cause serious damage to enzyme activity.Moreover, some specific substrates might also be sensitive to high O 2 concentrations.Likewise, if NOX has a high affinity toward O 2 with a low K MO , which could be achieved by enzyme engineering, air bubbling would be more suitable and efficient to supply O 2 for biocatalytic oxidation.
Based on the above, an operation map of the bubble column for gas bubbling is illustrated in Figure 7.The relationship between the O 2 concentration and protein concentration loss was also described; that is, protein concentration loss occurred less with N 2 bubbling and O 2 bubbling, but more protein concentration loss appeared with air bubbling.Meanwhile, three different areas were distinguished competitive effect area, bubbling area, and oxidation effect area (Figure 7).In the competitive effect area, N 2 could easily get into the O 2 channel of NOX, which caused fast enzyme deactivation (N 2 effect area).In the bubbling area, the loss of protein concentration occurred less compared with another two areas.In the oxidation effect area, the high O 2 concentration might cause over-oxidation of the amino acid residue, which was regarded as the reason for enzyme deactivation (O 2 effect area).The

Organic Process Research & Development
half-life of NOX generated in this study is also involved in this figure, and it is 2.2, 28.6, and 20.2 h under N 2 , air, and O 2 bubbling, respectively.The figure might possibly be a reference for the bubble column operation conditions.It indicated that the suggested O 2 concentration for the NOX reaction was in a range of 50−70%.In this range, the effects of N 2 (competitive effect) and O 2 (oxidation effect) were reduced, the protein concentration after bubbling was relatively high, and less protein loss occurred.Meanwhile, a higher oxygen concentration in the supplied gas was more efficient for oxygen mass transfer.However, it might have more requirements about gas flow control for different N 2 and O 2 ratios to get the suitable O 2 concentration.Further experiments could be conducted to determine more specific oxygen concentrations.The studies of enzymes and cofactors, which are involved in the cascade reaction together with NOX (e.g., oxidoreductases, natural and artificial cofactors), are also important to determine appropriate processes for biocatalytic oxidation.
Enzyme Structure Analysis.The NOX surface properties, including the electric potential and hydrophobicity/hydrophilicity, were analyzed by UCSF ChimeraX (Version 1.4) to further explain the results of NOX deactivation (Figure 8b,d).The cross section of the NOX active site is also shown in Figure 8c,e.The electric potential distribution of NOX showed that the NOX surface potential was more negatively charged (Figure 8b), but the active site was more positively charged according to the cross-sectional view of the active site (Figure 8c).It has been reported that the bubbles in aqueous solution carry a surface charge and are negatively charged due to excess OH -groups at the corresponding gas−liquid interface. 29herefore, a positively charged active site is more easily adsorbed on the gas−liquid interface, which might be the possible driving factor for NOX adsorption and the following aggregation at the gas−liquid interface.Then, the enzyme loses its activity due to the change of the three-dimensional structure and the shape of the active site.In Figure 8d, the surface of NOX was more hydrophilic.On the contrary, the hydrophobic region appeared in the inner part of NOX, especially in the active site (Figure 8c).This is also one of the factors that drive enzyme deactivation because of the hydrophobicity of the gas− liquid interface.

■ CONCLUSIONS
Although enzyme deactivation has been known for a long time, the effects of pH, temperature, and solvents on enzyme stability are often studied in quiescent conditions.There are few studies that focus on the operational stability of enzyme, such as exposure to the gas−liquid interface and on the kinetic deactivation process of enzyme during operation.In this study, the kinetic stability of NOX was studied in a bubble column with different gases bubbling for 60 h, and the deactivation process was detected from 100% activity to less than 20% residual activity.It was found that NOX unfolding and aggregation at the gas−liquid interface resulted in the pH increase.The protein concentration decreased upon exposure to the gas−liquid interface, with most aggregation occurring with air bubbling compared with N 2 and O 2 bubbling.A high O 2 concentration might cause over-oxidation of the amino acid residue.The deactivation of NOX under argon and N 2 bubbling were very fast and had similar decreasing trends.It was found that the dissolved N 2 in solution caused fast deactivation of NOX compared with the N 2 −water interface, which was confirmed by bubbling N 2 and O 2 alternately and also the comparison of the NOX specific activity in quiescent conditions and bubble column with N 2 , air, and O 2 bubbling.Furthermore, the air−water interface had less effect on enzyme activity but caused serious protein loss from solution.A range of 50−70% O 2 concentrations were suggested for NOX reaction operation because of the less protein concentration loss and long half-life of NOX according to the operation map of the bubble column generated in this study.
These results help our understanding of the kinetic deactivation process of NOX.According to this result, enzyme engineering might be inspired by the fact that only focusing on enhancing the affinity of the substrate and the thermal stability might not be enough.More attention should be paid to the kinetic stability of enzymes exposed to different operation conditions in biocatalytic oxidation process.

■Figure 1 .
Scheme 1. Biocatalytic Oxidation by a Dehydrogenase Coupled with In Situ Cofactor Regeneration Using Water-Forming NOX

Figure 2 .
Figure 2. Change of temperature, pH, and the dissolved oxygen concentration of NOX solution in the bubble column as N 2 (a), air (b), and O 2 (c) were supplied for gas bubbling.(d) pH change of deionized water after gas bubbling (control experiment); 25 °C, 200 mL of NOX solution, and 0.15 L min −1 gas flow rate.

Figure 5 .
Figure 5. Diagram and description of different gas−liquid interfaces and the dissolved gases' concentration (a).The residual activity of NOX under N 2 bubbling 6.5 h and the following O 2 bubbling (b) and the first O 2 bubbling 6.5 h and following N 2 bubbling (c).0.05 L min −1 gas flow rate, 200 mL NOX solution, and 25 °C.

Figure 7 .
Figure 7. Operation map of the bubble column with the relationship between O 2 concentration and protein aggregation, NOX half-life.Three areas were distinguished by the shadow and blank areas, and it showed the different effects of the NOX deactivation process.Competitive effect: N 2 concentration was much higher than the O 2 concentration, and N 2 could easily get into the O 2 channel of NOX (N 2 effect area); bubbling area, the protein concentration loss occurred less compared with another two areas; oxidation effect: the high O 2 concentration caused over-oxidation of the amino acid residue (O 2 effect area).

Figure 8 .
Figure 8. Surface property of water-forming NOX (PDB: 5VN0) with (a) 3D structure view, (b) electric potential distribution, and (c) corresponding cross-sectional view of the active site bound to NADH, (d) hydrophobic/hydrophilic distribution, and (e) corresponding crosssectional view of the active site bound to NADH.Diagrams generated using UCSF ChimeraX (Version 1.4).

Table 1 .
Solubility of N 2 and O 2 in Water and the Deactivation Kinetics of NOX under Different Gases Bubbling, 0.05 L min −1 Gas Flow Rate, 200 mL of NOX Solution, and 25 °C

■ ASSOCIATED CONTENT * sı Supporting Information The
Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.oprd.3c00095.Absorbance decreasing of NADPH solution catalyzed by NOX; the standard curve of protein concentration; data of the protein concentration change in quiescent condition and in a bubble column with N 2 , air, and O 2 bubbling; and the relationship of the NOX specific activity to activity and protein concentration under quiescent and bubbling conditions (PDF) Department of Chemical and Biochemical Engineering, Technical University of Denmark, 2800 Kgs.Lyngby, Denmark; orcid.org/0000-0002-7976-2483;Email: jw@kt.dtu.dk